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Methods in
Molecular Biology 2482

Guiomar Solanas · Patrick-Simon Welz Editors

Circadian
Regulation
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire Hatfield,
Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
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constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Circadian Regulation

Methods and Protocols

Edited by

Guiomar Solanas
Institute for Research in Biomedicine, Barcelona Institute for Science and Tech, Barcelona, Spain

Patrick-Simon Welz
Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain
Editors
Guiomar Solanas Patrick-Simon Welz
Institute for Research in Biomedicine Hospital del Mar Medical Research Institute (IMIM)
Barcelona Institute for Science and Tech Barcelona, Spain
Barcelona, Spain

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2248-3 ISBN 978-1-0716-2249-0 (eBook)
https://doi.org/10.1007/978-1-0716-2249-0
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2022
Chapter 3, 4, 8, and 17 are licensed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/). For further details see license information in the chapter.
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Preface

This book compiles a series of methods on several aspects of circadian research. These
methods are thoroughly explained for newcomers to the field and contain some of the latest
techniques for expert scientists to be updated. The reader will find protocols covering the
wide variety of daily rhythmic processes, using diverse model organisms, for the analysis of
circadian rhythms in the SCN and in peripheral organs, describing in vitro systems and in
silico methods.

Barcelona, Spain Guiomar Solanas

Patrick-Simon Welz

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Sleep Under Preindustrial Conditions: What We Can Learn from It . . . . . . . . . . . 1

Leandro Casiraghi and Horacio O. de la Iglesia
2 The Structure-Based Molecular-Docking Screen Against Core
Clock Proteins to Identify Small Molecules to Modulate the
Circadian Clock. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Seref Gul and Ibrahim Halil Kavakli
3 Analysis of Complex Circadian Time Series Data Using Wavelets . . . . . . . . . . . . . 35
Christoph Schmal, Gregor Mönke, and Adrián E. Granada
4 Mathematical Modeling in Circadian Rhythmicity . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Marta del Olmo, Saskia Grabe, and Hanspeter Herzel
5 Bioinformatics and Systems Biology of Circadian Rhythms:
BIO_CYCLE and CircadiOmics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Muntaha Samad, Forest Agostinelli, and Pierre Baldi
6 Cell-Based Phenotypic Screens to Discover Circadian
Clock-Modulating Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Megumi Hatori and Tsuyoshi Hirota
7 Methods for Assessing Circadian Rhythms and Cell Cycle in
Intestinal Enteroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Miri Park, Yuhui Cao, and Christian I. Hong
8 Using ALLIGATORs to Capture Circadian Bioluminescence. . . . . . . . . . . . . . . . . 125
Aiwei Zeng and John S. O’Neill
9 Studying Circadian Clock Entrainment by Hormonal Signals. . . . . . . . . . . . . . . . . 137
Violetta Pilorz, Iwona Olejniczak, and Henrik Oster
10 In Vitro Assays for Measuring Intercellular Coupling Among
Peripheral Circadian Oscillators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Anna-Marie Finger
11 Circadian Control of Transcriptional and Metabolic Rhythms
in Primary Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Sung Kook Chun and Selma Masri
12 Electrophysiology of the Suprachiasmatic Nucleus: Single-Unit
Recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Martha U. Gillette and Jennifer W. Mitchell
13 Anatomical Methods to Study the Suprachiasmatic Nucleus. . . . . . . . . . . . . . . . . . 191
Eric L. Bittman
14 Circadian Analysis of Rodent Locomotor Activity in Home Cages . . . . . . . . . . . . 211
Paul Petrus and Paolo Sassone-Corsi

vii
viii Contents

15 Recording of Diurnal Gene Expression in Peripheral Organs

of Mice Using the RT-Biolumicorder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Georgia Katsioudi, Alejandro Osorio-Forero, Flore Sinturel,
Claudia Hagedorn, Florian Kreppel, Ueli Schibler,
and David Gatfield
16 Isolation and Sorting of Epidermal Interfollicular Stem
Cells for the Study of Circadian Rhythms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Paloma Solá and Valentina M. Zinna
17 Detecting Circadian Rhythms in Human Red Blood Cells
by Dielectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Andrew D. Beale, Fatima H. Labeed, Stephen J. Kitcatt,
and John S. O’Neill
18 Measuring Circadian Neutrophil Infiltration in Tissues by
Paired Whole-Mount Tissue Clearing and Flow Cytometry . . . . . . . . . . . . . . . . . . 265
Tommaso Vicanolo, Andres Hidalgo, and Jose M. Adrover
19 In Vivo Imaging of Circadian NET Formation During Lung
Injury by Four-Dimensional Intravital Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Alejandra Aroca-Crevillén, Andres Hidalgo, and Jose M. Adrover
20 Real-Time Measurement of Energy Metabolism Over Circadian
Time Using Indirect Calorimetry-Enabled Metabolic Cages . . . . . . . . . . . . . . . . . 301
Kevin B. Koronowski and Paolo Sassone-Corsi
21 Untargeted and Targeted Circadian Metabolomics Using Liquid
Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
and Flow Injection-Electrospray Ionization-Tandem Mass
Spectrometry (FIA-ESI-MS/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Anna Artati, Cornelia Prehn, Dominik Lutter,
and Kenneth Allen Dyar
22 Time-Restricted Feeding and Caloric Restriction: Two
Feeding Regimens at the Crossroad of Metabolic and
Circadian Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Amandine Chaix
23 Chromatin Immunoprecipitation and Circadian Rhythms. . . . . . . . . . . . . . . . . . . . 341
Kenichiro Kinouchi, Kazutoshi Miyashita, and Hiroshi Itoh
24 Fluorescent Reporters for Studying Circadian Rhythms in
Drosophila melanogaster . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Kathyani Parasram, Daniela Bachetti, Vania Carmona-Alcocer,
and Phillip Karpowicz
25 Visualization of Mutant Aggregates from Clock Neurons
by Agarose Gel Electrophoresis (AGERA) in Drosophila melanogaster . . . . . . . . . 373
Laura Delfino, Susanna Campesan, Giorgio Fedele,
Edward W. Green, Flaviano Giorgini,
Charalambos P. Kyriacou, and Ezio Rosato
 Contents ix

26 Methods for Delivery of dsRNAi Against Canonical Clock

Genes and Immunocytodetection of Clock Proteins in Crustacea . . . . . . . . . . . . . 385
David C. Wilcockson, Lin Zhang, and Charalambos P. Kyriacou
27 In Vivo Bioluminescence Analyses of Circadian Rhythms in
Arabidopsis thaliana Using a Microplate Luminometer . . . . . . . . . . . . . . . . . . . . . . 395
Masaaki Okada and Paloma Mas

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Contributors

JOSE M. ADROVER • Area of Cell and Developmental Biology, Fundaci
on Centro Nacional de
Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, USA
FOREST AGOSTINELLI • Institute for Genomics and Bioinformatics, University of California,
Irvine, CA, USA; Department of Computer Science and Engineering, University of South
Carolina, Columbia, SC, USA
ALEJANDRA AROCA-CREVILLÉN • Area of Cell and Developmental Biology, Fundaci
on Centro
Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
ANNA ARTATI • Metabolomics and Proteomics Core Facility, Helmholtz Zentrum München,
German Research Center for Environmental Health (GmbH), Neuherberg, Germany
DANIELA BACHETTI • Department of Biomedical Sciences, University of Windsor, Windsor,
ON, Canada
PIERRE BALDI • Department of Computer Science, University of California Irvine, Irvine,
CA, USA; Institute for Genomics and Bioinformatics, University of California, Irvine,
CA, USA
ANDREW D. BEALE • MRC Laboratory of Molecular Biology, Cambridge, UK
ERIC L. BITTMAN • Department of Biology and Program in Neuroscience & Behavior,
University of Massachusetts, Amherst, MA, USA
SUSANNA CAMPESAN • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
YUHUI CAO • Department of Pharmacology and Systems Physiology, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
VANIA CARMONA-ALCOCER • Department of Biomedical Sciences, University of Windsor,
Windsor, ON, Canada
LEANDRO CASIRAGHI • Department of Biology, University of Washington, Seattle, WA, USA
AMANDINE CHAIX • Department of Nutrition and Integrative Physiology, University of Utah,
Salt Lake City, UT, USA
SUNG KOOK CHUN • Department of Biological Chemistry, Center for Epigenetics and
Metabolism, Chao Family Comprehensive Cancer Center, University of California, Irvine
(UCI), Irvine, CA, USA
LAURA DELFINO • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
KENNETH ALLEN DYAR • German Center for Diabetes Research (DZD), Neuherberg,
Germany; Metabolic Physiology, Institute for Diabetes and Cancer (IDC), Helmholtz
Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany
GIORGIO FEDELE • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
ANNA-MARIE FINGER • Charité Universit€ a tsmedizin Berlin, Berlin, Germany; Freie
Universit€ at Berlin, Berlin, Germany; Humboldt-Universit€ a t zu Berlin, Berlin, Germany;
Laboratory of Chronobiology, Berlin Institute of Health, Berlin, Germany
DAVID GATFIELD • Center for Integrative Genomics, University of Lausanne, Lausanne,
Switzerland

xi
xii Contributors

MARTHA U. GILLETTE • Department of Cell and Developmental Biology and Neuroscience

Program, Beckman Institute for Advanced Science and Technology, University of Illinois at
Urbana-Champaign, Urbana, IL, USA
FLAVIANO GIORGINI • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
SASKIA GRABE • Institute for Theoretical Biology, Charite and Humboldt Universit€a t zu
Berlin, Berlin, Germany
ADRIÁN E. GRANADA • Charité Comprehensive Cancer Center, Charité Universit€ atsmedizin
Berlin, Berlin, Germany; German Cancer Consortium (DKTK), German Cancer
Research Center (DKFZ), Heidelberg, Germany
EDWARD W. GREEN • German Cancer Research Center (DKFZ), Heidelberg, Germany
SEREF GUL • Chemical and Biological Engineering, Koç University, Istanbul, Turkey
CLAUDIA HAGEDORN • Biochemistry and Molecular Medicine, Center for Biomedical
Education and Research (ZBAF), Faculty of Health, Witten/Herdecke University, Witten,
Germany
MEGUMI HATORI • Institute of Transformative Bio-Molecules, Nagoya University, Nagoya,
Japan
HANSPETER HERZEL • Institute for Theoretical Biology, Charite and Humboldt Universit€ at
zu Berlin, Berlin, Germany
ANDRES HIDALGO • Area of Cell and Developmental Biology, Fundaci
on Centro Nacional de
Investigaciones Cardiovasculares (CNIC), Madrid, Spain
TSUYOSHI HIROTA • Institute of Transformative Bio-Molecules, Nagoya University, Nagoya,
Japan
CHRISTIAN I. HONG • Department of Pharmacology and Systems Physiology, University of
Cincinnati College of Medicine, Cincinnati, OH, USA
HORACIO O. DE LA IGLESIA • Department of Biology, University of Washington, Seattle, WA,
USA
HIROSHI ITOH • Department of Endocrinology, Metabolism, and Nephrology, Keio University
School of Medicine, Tokyo, Japan
PHILLIP KARPOWICZ • Department of Biomedical Sciences, University of Windsor, Windsor,
ON, Canada
GEORGIA KATSIOUDI • Center for Integrative Genomics, University of Lausanne, Lausanne,
Switzerland
IBRAHIM HALIL KAVAKLI • Chemical and Biological Engineering, Koç University, Istanbul,
Turkey; Molecular Biology and Genetics, Koç University, Istanbul, Turkey
KENICHIRO KINOUCHI • Department of Endocrinology, Metabolism, and Nephrology, Keio
University School of Medicine, Tokyo, Japan
STEPHEN J. KITCATT • Department of Mechanical Engineering Sciences, University of Surrey,
Surrey, UK
KEVIN B. KORONOWSKI • Center for Epigenetics and Metabolism, U1233 INSERM,
Department of Biological Chemistry, University of California, Irvine, CA, USA
FLORIAN KREPPEL • Biochemistry and Molecular Medicine, Center for Biomedical Education
and Research (ZBAF), Faculty of Health, Witten/Herdecke University, Witten, Germany
CHARALAMBOS P. KYRIACOU • Department of Genetics and Genome Biology, University of
Leicester, Leicester, UK
FATIMA H. LABEED • Department of Mechanical Engineering Sciences, University of Surrey,
Surrey, UK
 Contributors xiii

DOMINIK LUTTER • Computational Discovery Research, Institute for Diabetes and Obesity
(IDO), Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany;
German Center for Diabetes Research (DZD), Neuherberg, Germany
PALOMA MAS • Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-
UB, Barcelona, Spain; Consejo Superior de Investigaciones Cientı́ficas (CSIC), Barcelona,
Spain
SELMA MASRI • Department of Biological Chemistry, Center for Epigenetics and Metabolism,
Chao Family Comprehensive Cancer Center, University of California, Irvine (UCI),
Irvine, CA, USA
JENNIFER W. MITCHELL • Department of Cell and Developmental Biology and Neuroscience
Program, Beckman Institute for Advanced Science and Technology, University of Illinois at
Urbana-Champaign, Urbana, IL, USA
KAZUTOSHI MIYASHITA • Department of Endocrinology, Metabolism, and Nephrology, Keio
University School of Medicine, Tokyo, Japan
GREGOR MÖNKE • European Molecular Biology Laboratory, Heidelberg, Germany
JOHN S. O’NEILL • MRC Laboratory of Molecular Biology, Cambridge, UK
MASAAKI OKADA • Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-
UAB-UB, Barcelona, Spain
IWONA OLEJNICZAK • Institute of Neurobiology, Center of Brain, Behavior & Metabolism,
University of Lübeck, Lübeck, Germany
MARTA DEL OLMO • Institute for Theoretical Biology, Charite and Humboldt Universit€ at zu
Berlin, Berlin, Germany
ALEJANDRO OSORIO-FORERO • Department of Fundamental Neurosciences, University of
Lausanne, Lausanne, Switzerland
HENRIK OSTER • Institute of Neurobiology, Center of Brain, Behavior & Metabolism,
University of Lübeck, Lübeck, Germany
KATHYANI PARASRAM • Department of Biomedical Sciences, University of Windsor, Windsor,
ON, Canada
MIRI PARK • Department of Pharmacology and Systems Physiology, University of Cincinnati
College of Medicine, Cincinnati, OH, USA
PAUL PETRUS • Center for Epigenetics and Metabolism, U1233 INSERM, Department of
Biological Chemistry, University of California, Irvine, CA, USA
VIOLETTA PILORZ • Institute of Neurobiology, Center of Brain, Behavior & Metabolism,
University of Lübeck, Lübeck, Germany
CORNELIA PREHN • Metabolomics and Proteomics Core Facility, Helmholtz Zentrum
München, German Research Center for Environmental Health (GmbH), Neuherberg,
Germany
EZIO ROSATO • Department of Genetics and Genome Biology, University of Leicester,
Leicester, UK
MUNTAHA SAMAD • Department of Computer Science, University of California Irvine,
Irvine, CA, USA; Institute for Genomics and Bioinformatics, University of California,
Irvine, CA, USA
UELI SCHIBLER • Department of Molecular Biology, Faculty of Sciences, University of Geneva,
Geneva, Switzerland
CHRISTOPH SCHMAL • Institute for Theoretical Biology, Humboldt Universit€ a t zu Berlin,
Berlin, Germany
FLORE SINTUREL • Division of Endocrinology, Diabetes, Nutrition and Patient Education,
Department of Medicine, University Hospital of Geneva, Geneva, Switzerland;
xiv Contributors

Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva,

Geneva, Switzerland
PALOMA SOLÁ • Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute
of Science and Technology, Barcelona, Spain
TOMMASO VICANOLO • Area of Cell and Developmental Biology, Fundaci
on Centro Nacional
de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
DAVID C. WILCOCKSON • Institute of Biological, Environmental and Rural Sciences,
Aberystwyth University, Aberystwyth, UK
AIWEI ZENG • MRC Laboratory of Molecular Biology, Cambridge, UK
LIN ZHANG • Department of Genetics and Genome Biology, University of Leicester, Leicester,
UK
VALENTINA M. ZINNA • Institute for Research in Biomedicine (IRB Barcelona), Barcelona
Institute of Science and Technology, Barcelona, Spain
 Chapter 1

Sleep Under Preindustrial Conditions: What We Can Learn

from It
Leandro Casiraghi and Horacio O. de la Iglesia

Abstract
Human sleep is regulated by light in two fundamental ways: The light-dark (LD) cycle entrains a circadian
clock that in turn regulates sleep timing, and light per se can acutely inhibit sleep. Throughout evolution,
these sleep regulatory systems became highly sensitive to the effects of light and they can be affected by the
relatively low light intensities that are used indoors. Thus, postindustrial living conditions have created built
environments that have isolated humans from the natural LD cycle and exposed them to an artificial one
that can affect daily sleep timing. Studying indigenous communities that have differential access to
electricity, as well as communities living in highly urbanized areas, we and others have shown that human
access to artificial light has delayed the daily onset of sleep but has had a smaller effect on its offset, leading
to an overall reduction in sleep duration that is pervasive in modern societies. In this chapter we discuss
these studies, highlight their main findings, and point to their limitations.

Key words Sleep, Artificial light, Indigenous communities, Preindustrial societies, Modern societies

1 The Conquest of the Night

Life on earth evolved under the inescapable selective pressure of the

alternation between day and night. Human evolution was no
exception and collection of food by hunter-gatherer communities
was limited to the daytime while the night was a time to seek refuge
in a safe sleeping place. The transition to agricultural communities
created not only more sustainable food resources but also built
environments in which humans could shield themselves from pre-
dators, extreme weather, and other dangers associated with a
nomadic life. These built environments demanded the use of artifi-
cial light, which became a key resource particularly during the long
winter nights of higher latitudes.
However, it was not until the industrial revolution, and partic-
ularly until the use of electric light became more widespread, that
artificial light eliminated natural daylight as a condition for human

Guiomar Solanas and Patrick-Simon Welz (eds.), Circadian Regulation: Methods and Protocols,
Methods in Molecular Biology, vol. 2482, https://doi.org/10.1007/978-1-0716-2249-0_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

1
2 Leandro Casiraghi and Horacio O. de la Iglesia

activity. The second half of the twentieth century saw an exponen-

tial increase in electricity production and use, and a concomitant
exponential decrease in its cost, brightly lighting the indoor envi-
ronment irrespective of the solar day [1]. Labor, social life, and the
acquisition and transmission of knowledge could suddenly con-
tinue beyond the daytime, transforming nighttime from a time
for rest and sleep to a time during which activity could continue
at will. Natural dusk, which was the inescapable call to the end of
daily activity, was replaced by electric dusk, the timing of which
could be controlled freely. This manipulation of light and the
associated conquest of the night came at a cost, and a major
consequence has been the progressive change in the daily timing
of sleep as humans transitioned from agricultural, to industrial, to
post-industrial and highly urbanized communities. Whether and
how human sleep duration changed in the last century remains a
matter of debate; however, before the night was artificially lit, it
would have been hard to imagine the levels of activity and social
interaction that are now typical of any urban community after dusk.
A quick visual representation of the sleep and light exposure pat-
terns in a rural community without access to electricity and a highly
urbanized community clearly reveals the impact of the access to
electric light (Fig. 1).

Fig. 1 The access to electric light delays sleep timing. Clock plots displaying light exposure and actimetry-
based sleep timing. The plot on the left is from participants in a Toba/Qom community that has no access to
electricity. The plot on the right is from participants living in a highly urbanized community (Seattle, WA, USA)
and sampled during the weekend. Green bars represent the interquartile range for the first and last times that
individuals are exposed to a light intensity of 50 lux. Purple and blue bars represent the times of sleep onset
and offset, respectively. Note that in both cases sleep starts after the last exposure to 50 lux, but this occurs
much later relative to solar time in the highly urbanized community, in which the sleep onset occurs after solar
midnight. Gray and white in the pie represent the natural night and day, respectively. Time represents the local
clock time
 Sleep Under Preindustrial Conditions: What We Can Learn from It 3

2 Artificial Light and Its Impact on Sleep

The 24-h cycle of day and night represented an evolutionary selec-

tive pressure for the temporal distribution of physiological and
behavioral outputs. For some species it became adaptive to forage
and feed during the night whereas for others this was the time to
sleep. Although biological systems could have simply responded to
24-h cyclic events in the environment with specific behavioral and
physiological outcomes, it was likely more adaptive to be able to
predict these events before their arrival. As a consequence, circadian
clocks, namely biological mechanisms that oscillate autonomously
with a ~24-h period, were selected and are now a defining feature of
living systems. The main advantage of an endogenous clock is its
ability to predict cyclic events in the environment; for instance,
animals could seek insulation from the cold night before they
experienced low nocturnal temperatures. Importantly, circadian
clocks have periods that are not exactly 24 h and therefore need
to be entrained by environmental cycles. In any given habitat, cycles
that more reliably relay solar time are more likely to be selected as
proximate environmental factors to entrain the clock, and for vir-
tually all species the light-dark (LD) is the most prominent entrain-
ing cycle or zeitgeber.
A consequence of evolutionary selection of the LD cycle as the
principal zeitgeber is that circadian clocks and the biological
rhythms they sustain are extremely sensitive to the influence of
light. The human circadian system is no exception to this, and its
master circadian clock can be entrained by relatively low-intensity
light [2]. Humans, however, are different from other species in
their unique ability to control their own LD cycle, and this artificial
LD cycle can impact circadian rhythms, including the sleep-wake
cycle, in many ways.
First, light can directly affect the phase of circadian rhythms
through direct retinal projections to the suprachiasmatic nucleus
(SCN), which houses the master circadian clock of mammals
[3]. Light has an effect on the clock’s phase that depends on the
time at which the light stimulus reaches the clock [4]. Light during
the end of the night and early morning advances the phase of the
clock whereas light during the end of the evening and beginning of
the night delays the clock. Typically, humans use artificial light to
extend the end of the daytime instead of advancing its beginning.
In other words, most humans with access to electricity who can
choose their timing of activity will keep the lights on many hours
after dusk (up to 8 h in high latitudes during the winter) but will
rarely turn the lights on many hours before dawn. This asymmetric
use of artificial light leads to a net delay—relative to the natural LD
cycle—in the circadian clock and the rhythms it sustains, leading to
a later daily timing of sleep.
4 Leandro Casiraghi and Horacio O. de la Iglesia

Among the circadian rhythms that are consistently delayed is

the nocturnal release of melatonin, a hallmark of the biological
night. Melatonin is released by the pineal gland, and the circadian
timing of its release is regulated by the SCN. Under normal con-
ditions, the onset of melatonin occurs a few hours before bedtime,
and it represents an important cue to prepare the brain for the onset
of sleep. However, the signal is delayed by exposure to evening
artificial light.
Second, light can have acute effects on behavioral and physio-
logical rhythms. Brightly lit environments are particularly stimulat-
ing to humans. This is the consequence of the inhibitory effect that
light can have on sleep but also of the fact that light enables humans
to engage in different activities—TV watching, reading, social
interactions—that further inhibit sleep. Interestingly, light also
inhibits the nocturnal release of melatonin. The pineal releases
melatonin in a circadian fashion, but light also has the ability to
acutely reduce melatonin synthesis and release. Thus, light in the
late evening and early night will not only delay the clock but will
also abolish the release of the hormone that marks the beginning of
the night. The acute effects of light on sleep and melatonin release
push sleep onset to a later time in the night. Of note, these effects of
light have been exacerbated by the use of screens that have become
more widespread in the last two decades. A great part of human
evening activity is spent watching TV screens, monitors, or portable
devices, which are not only highly stimulating but themselves emit
light that is sufficient to elicit physiological effects [5].
A third and typically more ignored effect of artificial light is that
it allows humans to spend more time indoors. Most humans in
urbanized communities spend their working or school days within
artificially lit environments. Although these environments are
appropriately lit for indoor activities, they expose humans to light
levels that are typically an order of magnitude lower than natural
daylight. This shielding from natural daylight reduces the ability of
natural light to entrain circadian rhythms, which are therefore more
amenable to be entrained by mistimed artificial light.
All these effects of artificial light act together to delay human
sleep onset relative to what the sleep onset would be under natural
light conditions. This has been shown both by field and interven-
tion studies that have compared human sleep in the absence and
presence of artificial sleep. Notably, humans in highly urbanized
communities still wake up around sunrise to go to work or school,
and it is reasonable to predict that a trend to later sleep start
without delaying its end should shorten daily sleep. However,
whether daily sleep duration has historically diminished is still a
matter of controversy, one we may never fully elucidate. After all,
objective measures of sleep have only been available many decades
after electric light became popular. Nevertheless, it is clear that a
large proportion of humans living in modern cities sleep less than
 Sleep Under Preindustrial Conditions: What We Can Learn from It 5

what is considered necessary to sustain good health, and this

shorter than needed sleep would be much harder to achieve with-
out access to electric light.

3 Methods to Study the Effects of Electric Light on Human Sleep

The study of human circadian rhythms, including the sleep-wake

cycle, has been approached through a wide range of methods.
Isolation studies to demonstrate the endogenous nature of circa-
dian rhythms were first done in natural caves and later in artificial
isolation labs, which allowed for the removal of light cues, social
interactions, and other environmental variables that could provide
information about external time [6, 7]. Initially, these studies sug-
gested that the human circadian system was more sensitive to social
cues than environmental light cues, but later studies clearly estab-
lished that light could act as a strong zeitgeber in humans
[8, 9]. Furthermore, studies under carefully controlled laboratory
conditions were critical in demonstrating that electric light was
effective in resetting circadian rhythms even at relatively low inten-
sities used in indoor environments [2]. These studies, however,
could not assess the direct impact of electric light on human circa-
dian rhythms under field conditions. The fact that a single light
pulse of a given intensity can lead to a reliable phase shift of a given
rhythm in the lab does not mean the same pulse will elicit the same
shift in subjects living their normal life and exposed to other sources
of light throughout the day.
Field experiments are more appropriate to determine the real
impact of artificial light on real-life circadian physiology. However,
they obviously present limitations in terms of the physiological
variables that can be measured. These variables not only provide a
more holistic view of circadian outputs but in some cases, they are
critical to elucidate the mechanisms behind the changes in the
timing of behavioral rhythms. As described above, the same delay
on the onset of sleep could result from an acute effect of evening
light on behavior or from an effect of evening light on the phase of
the circadian clock. In either case, the behavioral expression of the
effect of light is the same, i.e., a later sleep start, but the mechanism
behind each case is radically different. To distinguish between
them, a more reliable marker of circadian phase such as the
dim-light melatonin onset (DLMO) is needed [10]. The DLMO
can be determined through serial blood or saliva samples. However,
a limitation is that the samples need to be taken under dim-light,
which is sometimes difficult or not possible under field conditions.
The ideal field experiment to assess the impact of electric light
on human circadian rhythms would be a longitudinal experiment in
which a group of subjects living in the same place gain or lose access
to electricity and their rhythms are monitored before and after the
6 Leandro Casiraghi and Horacio O. de la Iglesia

introduction of electric light. Such a scenario is difficult to come by,

but a similar longitudinal approach was used by Wright and collea-
gues [11]. The team monitored their own actimetry-based sleep
and DLMO in their typical urban setting during the summer, and
later monitored the same rhythms during a camping trip in which
they used no artificial sources of light. They found that in the
presence of electricity sleep timing was delayed, and this delay was
consistent with a later daily melatonin rhythm release, clearly indi-
cating a direct effect of electric light on the phase of the circadian
system. Although this study did not show a shortening of daily
sleep duration, the team performed a similar study during the
winter and found that sleep onset was advanced during winter
camping without a change in sleep offset, leading to a lengthening
of daily sleep duration [12].
In most field scenarios longitudinal studies are not feasible, and
investigators have gravitated toward communities with limited or
no access to electricity to assess sleep timing. Actimetry represents
the gold standard to determine sleep timing in field experiments
and in some groups has been shown to be much more accurate than
diary-based self-assessment of sleep [13]. Although wrist actimetry
has been validated with polysomnographic recordings as a reliable
estimate of sleep timing, it does not allow for the determination of
sleep stages [14–16]. Thus, actimetry alone provides little informa-
tion on the physiological basis of changes in sleep parameters, but it
does provide an accurate reflection of how the timing of sleep
changes under different environmental conditions.
A major limitation of field studies of communities living under
preindustrial conditions is the lack of appropriate control groups. It
is not hard to find communities still living under rural conditions
and even without access to electricity, but it is usually much harder
to find communities that can serve as controls. Ideally, commu-
nities that are ethnically and culturally similar but do have access to
electricity or live in more urbanized communities should be used as
comparison groups.

4 Lessons from Sleep Under Preindustrial Conditions

Until recently, studies assessing sleep timing in rural communities

and, more specifically, communities without access to electricity
were based on diaries on which participants self-assessed their
times of sleep onset and offsets. Although sleep diaries can provide
an accurate reflection of sleep timing under well-controlled condi-
tions, they are less reliable in long-term studies where investigators
cannot assure daily compliance, and in communities with low liter-
acy levels. Furthermore, they may be particularly less informative in
communities in which activities follow no strict clock time.
Sleep Under Preindustrial Conditions: What We Can Learn from It 7

Nevertheless, early as well as more recent studies based on data

from diaries and interviews have provided valuable information
about sleep in different populations around the world (Table 1).
Worthman and Brown [17] studied Egyptian villagers and found
that sleeping arrangements, and particularly cosleeping, which was
highly prevalent in this population, affected the timing and quality
of sleep. Beckinschtein and collaborators [18] followed a native
Mapuche community in the Argentinian Patagonia and described
a clear seasonal difference in the timing of sleep which was also tied
to a seasonal migratory pattern. Louzada and Menna-Barreto [19]
compared the sleep habits of adolescents in a rural area in Southern
Brazil and found that those with access to electricity had later slept
times, and that the presence of a TV in their house correlated with
later bedtimes. Even in studies that use actimetry, diaries and inter-
views provide invaluable contextual information. For instance,
Yetish et al. [20] report that neither the San people in Namibia
nor the Tsimane people in Bolivia have a word for “insomnia”—a
pretty telling observation. Most importantly, these studies as a
whole underscore that we cannot simply frame sleep studies on
whether people live under preindustrial conditions and have no
access to electric light; sleep can be affected vastly by factors includ-
ing geography and weather, cultural traits, familial composition,
and socioeconomic status.
The amenability of wrist activity monitors provided a noninva-
sive, objective, and quantitative method to determine the time of
sleep onset and offset with high accuracy. These devices can also
measure light exposure as well as other physiological variables and
have become an invaluable tool for field sleep studies in remote
areas. In the last few years, multiple research teams have used wrist
activity as a powerful tool to objectively measure sleep timing, and
in many cases, to demystify assumptions about sleep under prein-
dustrial conditions. Indeed, studies have nothing but proved that
there is no normative sleep timing. A first pioneering study using
this technology showed sleep patterns in members of a small com-
munity living without electricity in a small island near mainland
New Guinea [21]. Although the study lacked a control group
(urban, with electricity), the authors made truly interesting find-
ings. For instance, whereas wake-up time was highly synchronous
among the study participants, their sleep onset was much more
variable, both within and between individuals. This result contrasts
with the very low variability in both sleep onset and offset that we
found in the Toba/Qom people living without electricity (Fig. 1;
[22]). Similarly, whereas the access to electric light is typically
associated with a delayed sleep onset, it was shown to shorten
sleep in some communities but not others (Table 1). Furthermore,
although fragmented sleep and chronotype variability seem to be
the norm and may represent an adaptive trait in Hadza hunter-
Table 1
8

Summary of studies classified by the population studied and the research team or teams. When more than one work is referenced for the same
population and team, methods and main findings are summarized indistinctly for the sake of simplicity. Rows are ordered chronologically

Population/
community Region Type of study Methodology Main findings References
Trobriand Tauwema Observational, population with Actimetry Young infants displayed ultradian sleep [21]
islanders Village, Papúa no access to electricity only patterns and slept more. Wives in couples
Nueva Guinea slept more than their husbands.
Mapuche Neuquén, Observational, population with Sleep diaries Wake-up times are delayed in the winter. [18]
Argentina no access to electricity only
Rural village Paraná State, Controlled, access to electric Questionnaires, Adolescents with no access to electric light [19, 33, 34]
adolescents Brazil light vs. no access; different actimetry, slept more and showed no sleep deprivation.
school times DLMO School times correlated with sleep duration
and onset. Access to electric light correlated
with delayed phase of sleep.
Egyptian villagers El Cairo, Egypt Observational Diaries Cosleeping correlated with less sleep but of [17, 35]
better quality. High occurrence of daytime
Leandro Casiraghi and Horacio O. de la Iglesia

napping.
Haitian rural Fondwa, Haiti Observational, population with Actimetry Sleep fragmentation decreased with age. [36]
villagers no access to electricity only Longer times of staying-in-bed than in
industrialized societies, but similar effective
sleep duration.
Toba/Qom Formosa, Controlled, different levels of Actimetry, diaries, Access to electricity correlated with delayed [22, 24, 25]
Argentina access to electric light and DLMO sleep onset and reduced sleep duration.
urbanity Larger differences between electricity levels in
the winter.
Electric light associated with delayed onset of
melatonin release during the winter.
Amazonian Amazonian Controlled, access to electric Diaries, actimetry, Access to electricity and urbanization [26, 37, 38]
rubber tappers Extractive light vs. no access DLMO correlated with delayed and shorter sleep.
Reserve, Brazil
 San people Northeastern Mixeda, communities with no Actimetry, Shorter and later sleep in the summer. Low [20]
Namibia access to electric light interviews incidence of insomnia.
Tsimane people Beni Mixeda, communities with no Actimetry, Shorter and later sleep in the summer. Low [20]
Department, access to electric light interviews incidence of insomnia.
Bolivia
Hadza people Northern Observational, mixeda Actimetry, Short sleep duration compared to industrial [20, 23, 39]
Tanzania interviews societies. Communal “sentinel” pattern of
sleep.
Rural villagers Baependi, Brazil Observational Actimetry, DLMO, One of the earliest chronotypes in literature. [40, 41]
questionnaires Women slept more than men.
Malagasy rural Mandena, Observational, population with Actimetry, PSG High prevalence of “segmented” night sleep [42]
villagers Madagascar no access to electric light and daytime napping. Short sleep duration
only compared to industrial societies.
Milange and Zambézia, Controlled, urban vs. rural Actimetry Urbanity was associated with delayed sleep [28]
Tengua Mozambique conditions onset. No differences in sleep duration.
communities
Quilombolas Southern Brazil Controlled, different levels of Actimetry, MCT Access to electricity and urbanization [27]
access to electric light and Questionnaire correlated with delayed and shorter sleep.
urbanity
Indigenous Tanna Island, Controlled, access to electric Actimetry Access to electricity associated with sleep onset [29]
Melanesians Vanuatu light vs. no access and reduces sleep duration and efficiency.
a
Yetish et al. [20] compares these three preindustrial populations
Sleep Under Preindustrial Conditions: What We Can Learn from It
9
10 Leandro Casiraghi and Horacio O. de la Iglesia

gatherers, >8 h consolidated sleep with no chronotype variation

and no naps appears to be the norm for the Toba/Qom people
[22, 23].
Beyond the temporal distribution of sleep, total daily sleep
duration in preindustrial communities—under the assumption
that it could represent some indicator of a minimal need for daily
sleep—has received particular attention. Although there is nothing
normative about preindustrial sleep (see Subheading 5) it is impor-
tant to highlight that total daily sleep is equally variable among
communities living in rural areas without electricity. Several studies
confirmed the long-held intuition that access to electricity delays
sleep onset, even when involving communities that differ ethnically,
geographically, and culturally (Table 1): Toba/Qom communities
in the north of Argentina [22, 24, 25], Amazonian workers [26],
Quilombola natives in Brazil [27], Milange and Tengua commu-
nities in Mozambique [28], or indigenous Melanesians in Vanuatu
[29]. Importantly, the delay in sleep onset associated with access to
electric light shortened sleep duration in some of these commu-
nities but not in others, suggesting that changes in sleep duration
may be associated with different trade-offs in different commu-
nities. This is further underscored by the wide variability in sleep
duration under preindustrial conditions, which can range from
6.25 h (e.g., the Tsimane people in Bolivia) to 8.3 h (the Toba/
Qom people in Argentina) ([20, 25, 30]; Fig. 1). Interestingly,
even when the total sleep times vary widely among these groups,
they all show seasonal differences, with a strikingly similar length-
ening of sleep during the winter, although the mechanisms behind
this longer winter sleep may differ.
The access to electricity is not only associated with the amena-
bility of electric light but also with the access to other light-emitting
devices like TVs and screens from computers, tablets, and cell
phones. Furthermore, electric light allows for evening social activ-
ity, which together with these devices promotes a later bedtime.
This has raised the question of whether the delayed sleep associated
with access to electricity can directly be attributed to light or
depends on other factors associated with urban settings. By asses-
sing the DLMO in communities with and without electricity, we
and others have shown that, at least in part, the delayed sleep timing
is a consequence of a delayed circadian clock [24, 26].

5 Limitations of Preindustrial Sleep Studies

It is tempting to interpret sleep patterns found in communities

living in preindustrial conditions as a proxy of human ancestral
sleep. However, in the same manner in which no extant species
can be seen as basal or ancestral, sleep patterns we observe today,
regardless of the environmental conditions in which they occur, do
 Sleep Under Preindustrial Conditions: What We Can Learn from It 11

not necessarily reflect how humans slept historically or prehistori-

cally. It is important to keep in mind that over 300,000 years of
evolution separate modern humans from the first humans that
appear in Africa [31].
Several authors have also suggested sleep patterns under
non-industrial conditions are normative for sleep in humans living
in highly urban environments. The rationale is that sleep under
more “natural” conditions should reflect how much and what
kind of sleep we need under modern living conditions. This premise
is flawed for several reasons. First, there is really not such a thing as
“natural sleeping habitat.” The multidimensional nature of sleep
function determines that each environment demands a specific
timing and quality of sleep. In the same way that different prein-
dustrial environments demand different sleep patterns, highly urba-
nized environments likely demand drastically different sleep
behavior. In other words, the natural sleeping environment for a
person living in New York City is New York City and not some
remote place isolated from urban development.
Second, sleep timing and the manifestation of specific sleep
stages are not simply a consequence of the sleep needs that a specific
environment demands but also of the sleep that environment per-
mits. In other words, members of a community living under pre-
industrial conditions could also be subjected to chronic or acute
sleep deprivation because of environmental constraints.
Third, the possibility exists that original ethnic communities
from different continents differ in sleep genetic traits [32]. It is
conceivable that as Homo sapiens migrated from Africa to Australia,
Europe, Asia, and eventually the Americas, specific environments
selected for specific genetically determined sleep traits. For
instance, the Americas are believed to have been colonized by
migrations that reached the new continent through the Bering
Strait about 15,000 years ago, when the temperatures were consid-
erably lower than now. This environment likely demanded sleep
patterns that differed from those on the African continent. Further-
more, human colonization of new continents could have created
evolutionary bottlenecks for genetic sleep traits, which could
reduce the adaptability of sleep patterns to new environments.

6 Conclusions

The last two decades have seen a marked increase in studies that
have capitalized on the access to communities living in a range of
preindustrial conditions—from hunting-gathering to agricultural
peoples—to gain insight into sleep patterns in the absence of
urbanization. These studies have provided unequivocal evidence
that urbanization, and its associated increased exposure to electric
light, delays daily sleep timing and, in many cases, shortens its
12 Leandro Casiraghi and Horacio O. de la Iglesia

duration. These effects on sleep parameters are, in some cases,

mediated in part by a delay on the circadian system. Collectively,
these studies have confirmed that sleep under preindustrial condi-
tions can vary tremendously both in duration and temporal distri-
bution, corroborating the plasticity of sleep as a behavioral trait and
underscoring that sleep under low levels of urbanization should not
be taken as a normative sleep for other human communities.

Acknowledgments

We are thankful to Isabelle Hua and Gideon Dunster for their help
with Fig. 1, and to Dr. Ray Sanchez for comments on the original
manuscript. Supported by NSF RAPID award #1743364 and Lea-
key Foundation grant #1266 to HOD.

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 Chapter 2

The Structure-Based Molecular-Docking Screen Against

Core Clock Proteins to Identify Small Molecules to Modulate
the Circadian Clock
Seref Gul and Ibrahim Halil Kavakli

Abstract
Circadian rhythms are part of the body’s clock, which regulates several physiological and biochemical
variables according to the 24-h cycle. Ample evidence indicated disturbance of the circadian clock leads to
an increased susceptibility to several diseases. Therefore, a great effort has been made to find small
molecules that regulate circadian rhythm by high-throughput methods. Having crystal structures of core
clock proteins, makes them amenable to structure-based drug design studies. Here, we describe virtual
screening methods that can be utilized for the identification of small molecules regulating the activity of
core clock protein Cryptochrome 1.

Key words Circadian rhythm, Virtual screening, Cryptochrome, Docking, Drug discovery

1 Introduction

The circadian rhythms are endogenous autonomous oscillators,

which enable organisms to adapt their physiological variables to
24-h day/night cycles [1]. They are generated by a mechanism
known as the circadian clock, which modulates memory, blood
pressure, immune responses, etc. by a periodic transcriptional reg-
ulation [2–4]. The cellular clockwork is generated by positive and
negative transcriptional and translational feedback loops [5, 6]. In
mammals, the positive loop consists of two core clock proteins,
BMAL1 and CLOCK. These proteins form heterodimers and bind
to E-BOX elements (CACGTG) in the Period (Per), Cryptochrome
(Cry) and clock control genes. PER and CRY along with casein
kinase Iε (CKIε) form trimeric structure and translocate to the
nucleus where it represses BMAL1/CLOCK-driven transcription.
Upon phosphorylation, CRYs are ubiquitinated by E3 ubiquitin

Guiomar Solanas and Patrick-Simon Welz (eds.), Circadian Regulation: Methods and Protocols,
Methods in Molecular Biology, vol. 2482, https://doi.org/10.1007/978-1-0716-2249-0_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

15
16 Seref Gul and Ibrahim Halil Kavakli

ligases, e.g., FBXL3 and FBXL21, and directed to the proteasome

for degradation. FBXL3 and FBXL21 bind to the primary (FAD
binding) pocket of CRYs and act antagonistically and differentially
on their stability in the cytosol and the nucleus [7, 8]. This core
clock machinery drives rhythmic output called clock-controlled
genes in almost every tissue. Indeed, 50% of detected metabolites
are under circadian control in mouse liver [9] and nearly 43% of all
protein coding genes are under the control of the circadian clock in
at least one tissue [10]. A recent study highlights the importance of
a post-translational switch that modulates the activity of the
cystathionine ß-synthase by CRY1 to regulate amino acid
metabolism [11].
Epidemiological and genetic studies have indicated that circa-
dian disturbances are associated with increased risks of different
types of diseases. A defect in PER2 produces familial advanced
sleep phase syndrome [12, 13]; an analogous mutation causes the
same phenotype in mice [14]. People with a causal mutation in
CSNK1D and an associated variant in CSNK1E display advanced
sleep phase syndrome and delayed sleep phase syndrome, respec-
tively [15, 16]. A human CLOCK variant is associated with diurnal
sleep preference [17]. Circadian core clock genes are also associated
with a host of neurological disorders including schizophrenia [18–
21], unipolar major depression [22], and bipolar disorder
[23, 24]. Another study leads to an identification of an SNP in
DEC2 in short sleeper patients [25]. A human gain-of-function
CRY1 variant (exon 11 skipping mutation in C-tail of CRY1), and
another CRY1 mutant (exon 6 skipping mutation in PHR of
CRY1) were found in people suffering from familial delayed sleep
phase disorder and attention deficit/hyperactivity disorder
[26, 27]. The importance of a robust circadian clock for health is
increasingly recognized, and therefore the identification of mole-
cules that modulate the circadian clock became a hot topic. The
high-throughput screening was instrumental for identifying the
molecules affecting the circadian clock [28–31]. However, that
approach is expensive and slow, to find specific molecules for core
clock proteins. To identify a molecule library that regulates the core
clock proteins (PER, BMAL1, CLOCK, and CRY) is currently a
scientific challenge. With the recent reports of resolved crystal
structures of core clock proteins and their interacting partners
(CRY-FBXL3 (pdbID: 4K0R), BMAL1-CLOCK (pdbID: 4F3L),
and CRY-PER (pdbID: 4U8H)), now it is possible to perform in
silico analysis against them. A recent study with in silico methods
was able to identify a molecule (CLK8) to target the CLOCK
protein [32]. Further in vivo and in vitro studies revealed that
CLK8 regulates CLOCK/BMAL1 entry into the nucleus and reg-
ulates the circadian rhythm.
 Structure-Based Drug Discovery for Clock Proteins 17

2 Materials

All of the programs listed below can be executed in Linux-based

computers. Researchers who want to use these procedures should
have at least a beginner level of Linux computer usage.

2.1 Homology If the protein does not have the crystal structure, homology mod-
Modeling Server eling tools should be used to construct 3D structure. Similarly,
missing atoms of the crystal structures can be added with the
same approach. Freely available and user-friendly homology mod-
eling servers can be used: RaptorX (http://raptorx.uchicago.edu/
StructPredV2/predict/), Swiss-Model (https://swissmodel.
expasy.org/interactive), and Phyre (http://www.sbg.bio.ic.ac.uk/
~phyre2/html/page.cgi?id¼index).

2.2 Programs to Pymol, Chimera, and VMD programs can be used to visualize and
Visualize Protein manipulate protein structures. Manual and tutorial of these pro-
Structures grams are available online to perform various operations such as
visualization, mutation, rotamer selection, and structure prepara-
tion for molecular dynamics (MD) simulations. Chimera and VMD
are free of charge for academic users with registration requirements
through their websites (https://www.cgl.ucsf.edu/chimera/down
load.html, https://www.ks.uiuc.edu/Development/Download/
download.cgi?PackageName¼VMD). Pymol offers an education
version for free and an alternatively open-source version can be
installed (https://pymol.org/dokuwiki/?id¼installation). The
usage of these programs will be mentioned in relevant sections.

2.3 Retrieving Crystal structures of CRY1 (PDB ID: 4K0R, 5T5X, or 6KX4) can
Protein Structures be downloaded from the protein data bank [33–35]. 4K0R is the
from Protein Data first resolved photolyase homology region (PHR) of mouse CRY1
Bank (PDB) structure with 2.65 Å in 2013 [33]. Higher-resolution PHR
domain of CRY1 structures with 1.84 and 2.00 Å were reported
recently [34, 35].

2.4 NAMD Program NAMD software is required to run MD simulations of protein.

and CHARMM Force Depending on the computer power, various versions of NAMD
Field have been developed and can be freely downloaded by academic
users (https://www.ks.uiuc.edu/Development/Download/down
load.cgi?PackageName¼NAMD). To simulate biological macro-
molecules, we need to calculate forces between atoms within a
molecule and between molecules. Thus, to describe these forces,
set of parameters that are generated after extensive benchmarking
studies, named force fields, are used. Ample high-quality force
fields are available in the literature and can be readily used with
NAMD. Since we used the CHARMM force field in our studies we
18 Seref Gul and Ibrahim Halil Kavakli

suggest using the latest version of CHARMM (http://www.

charmm-gui.org/?doc¼toppar, https://www.charmm.org/
charmm/resources/charmm-force-fields/#charmm).

2.5 Small Molecules Commercially available small molecule (SM) libraries are available
in sdf format with 2D structural information. Since 3D structural
information and partial charges of atoms in molecules are required
for docking simulations, sdf format should be converted to pdbqt
format.

2.6 Autodock Vina Autodock Tools (http://autodock.scripps.edu/resources/adt) and

and Autodock Tools Autodock Vina (http://vina.scripps.edu/download.html) are used
to prepare SMs and dock them to the receptor, respectively. Open-
babel can convert sdf format to pdb format, then Autodock Tools is
used to get pdbqt format of molecules from pdb files. In addition to
SM conversion, Autodock Tools is also used to prepare the pdbqt
format of the receptor (protein) and to determine the coordinates
of the docking site on the receptor. Vina is used for docking
simulations of SMs to the target pocket of receptors. At the end
of the docking simulations, Vina generates the binding mode of the
molecule in the pocket and calculates Vina binding energy between
molecule and receptor. The Autodock Tools program is used to
visualize and analyze docking results.

3 Methods

3.1 MD Simulation Two approaches are utilized for docking simulations in the litera-
ture: (a) docking molecules to the crystal structure of the receptor
and (b) docking molecules to the structure of the receptor equili-
brated under physiological conditions. We are not going to com-
pare the advantages and disadvantages of these approaches in this
chapter, but simply we prefer to use equilibrated receptor structures
in our docking simulations. Before starting, generate a new folder
named “structurebased,” download and save the below-mentioned
files into this folder.

3.1.1 Homology Since all crystal structures of CRY1 have missing atoms, homology
Modeling of CRY1 modeling should be performed. Swiss-Model server can be used to
get the full-length CRY1-PHR structure. The protein sequence of
mouse CRY1 can be retrieved from NCBI data bank.
1. Go to the website of NCBI (https://www.ncbi.nlm.nih.gov/),
select “Protein” from the drop-down menu and search for
“AAD39548.1”.
2. Click “FASTA” to get the fasta format of the CRY1 amino acid
sequence.
 Structure-Based Drug Discovery for Clock Proteins 19

3. Copy and paste sequence of CRY1 to SWISS-MODEL server

(https://swissmodel.expasy.org/interactive) (Fig. 1a) (see
Note 1).
4. Clicking “Search For Templates” will search for the crystal
structures having the highest identity and similarity to the
query sequence.
5. Click 5t5x that has the highest resolution among available
structures (Fig. 1b) (see Note 2).
6. Let’s choose 5t5x that has the highest coverage, identity, and
resolution among available structures. Then click “Build Mod-
els.” Download the PDB file by clicking
Model > PDB Format > save as CRY1.pdb (Fig. 1c) (see
Note 3).

3.1.2 Preparing CRY1 for 1. To assign protonation states of residues by using PROPKA
MD Simulation server (http://server.poissonboltzmann.org/pdb2pqr) (see
Note 4).
(a) Click “Upload a PDB file” to upload CRY1.PDB.
(b) Select “Use PROPKA to assign protonation states at
provided pH.”
(c) Forcefield to use: CHARMM.
(d) Output naming scheme to use: CHARMM.
(e) In “Additional Options” check the following: “Ensure
that new atoms are not rebuilt too close to existing
atoms,” “Optimize the hydrogen bonding network,”
and “Add/keep chain IDs in the PQR file.”
(f) Download output “jobid.pqr”. Rename file as CRY1_p.
pqr.
(g) Open CRY1_p.pqr with PyMol, first open Pymol, then
File > Open > (browse molecule) > Open
File menu > Save molecule > check “one file” and
“global” and click OK > CRY1_p.pdb (into the “struc-
turebased” folder)
2. To solvate protein and add an appropriate concentration of salt
we are going to use VMD.
(a) Download top_all36_prot.rtf into the “structurebased”
folder from http://www.charmm-gui.org/?doc¼toppar
(see Note 5).
(b) First, we need to generate psf file. Generate a text file
“psfgen.in” and type commands given in Subheading 4.
Open VMD in the same directory with CRY1_p.pdb and
the topology file (in “structurebased” folder). In the
“VMD Main” panel
20 Seref Gul and Ibrahim Halil Kavakli

Fig. 1 Generating homology model of CRY1 using SWISS-MODEL server. (a) Query page, (b) template selection
page, (c) model page
 Structure-Based Drug Discovery for Clock Proteins 21

Fig. 2 Snapshot from VMD session generated after CRY1 solvation

Extension menu > Tk console: source psfgen.in (see

Note 6).
(c) Protein will be solvated and NaCl will be added. Similar to
the previous step generate a text file “solvation.in” and
type the following to Tk console:

resetpsf
mol delete all
source solvation.in

At the end of this step ionized.pdb and ionized.psf

files will be produced (see Note 7) (Fig. 2).
3. Next, the dimension of system should be measured and gener-
ate pdb file with restrained Cα atoms that is required for
running the MD simulations.
(a) Type the following on the Tk console (without deleting
anything from the previous step):

set everyatom [atomselect top all]

measure minmax $everyatom (see Note 8)

(b) In MD simulation the system should be gradually heated.

Thus, Cα atoms of all residues are restrained and let side
chains of amino acids to move by sequential heating. At
22 Seref Gul and Ibrahim Halil Kavakli

this step, we are going to set up a pdb file (CA_rest.pdb)

containing restrained Cα atoms.
Start a new VMD session:
File > New Molecule > Browse > ionized.psf.
Right click on ionized.psf (on VMD Main) > Load
Data onto Molecule > Browse > ionized.
pdb > OK > Load
Type the following to Tk console: source restrain.in
(see Note 9).

3.1.3 Running MD Before running the simulation, minimization of the system should
Simulation be performed. Then gradually heat and equilibrate the system.
Finally, we are going to use coordinates, velocity, and extended
system configuration files of minimized, heated, and equilibrated
system to run the MD simulation.
1. Following parameter files should be downloaded to working
directory (into the “structurebased” folder): par_all36_prot.
prm, par_all36_lipid.prm, par_all36_carb.prm, par_al-
l36_cgenff.prm from (http://www.charmm-gui.org/?
doc¼toppar) and toppar_water_ions_namd.str from (http://
mackerell.umar yland.edu/~kenno/cgenf f/program.
php#namd). The last file is the edited version of toppar_wa-
ter_ions.str to be able to use it in NAMD.
2. Make four folders and name them as Minimized, Heat, Equi-
librium, and Production. Next move parameter files given in
supplementary materials minimization.in, heating.in, equilib-
rium.in, production.in to corresponding folders.
3. Go to “Minimize” folder and run minimize.in via NAMD by
typing the following:

~/namd/namd2 +p8 minimize.in > CRY1_minimize.log (see Note

10)

4. After successful completion of the minimization, go to “Heat-

ing” folder and run the following:

~/namd/namd2 +p8 heating.in > CRY1_heating.log (see Note 11)

Upon completion of the heating, go to “Equilibrium”

folder and run the following:

~/namd/namd2 +p8 equilibrium.in > CRY1_equilibrium.log

Upon completion of the equilibration, go to “Production”

folder and run the following:

~/namd/namd2 +p8 production.in > CRY1_production.log

 Structure-Based Drug Discovery for Clock Proteins 23

5. The equilibriated frame of the production run can be used for

docking simulations. To extract the last frame, go to “Produc-
tion” file and run VMD then:
File > New Molecule > Browse > ionized.
psf > Load > Browse > CRY1_production.dcd > Load
Then select (click on) ionized.psf on the VMD Main
panel and
File > Save Coordinates
From the Save Trajectory panel type protein into Selected
atoms section,
Type the last step in the “First” and “Last” sec-
tions > Save > CRY1.pdb
6. Now we have CRY1 structure equilibrated under physiological
conditions and ready to be used for the docking.

3.2 Docking Determining the target pocket, preparing molecules and receptor
(s) in pdbqt format are crucial steps in docking studies. Below we
are going to describe these steps for CRY1. Before going into
details generate a folder named “docking.” Move CRY1.pdb file
to the docking folder.

3.2.1 Preparation of 1. Open Autodock Tools program from the terminal, e.g., ~/
CRY1 for Docking MGLTools-1.5.6/MGLToolsPckgs/AutodockTools then
Simulations File > Rad Molecule > CRY1.pdb
Grid > Macromolecule > Choose > CRY1 > Select
molecule
New panel should appear: type CRY1.pdbqt > Save (see
Note 12).
2. A grid center point and box size should be determined for
docking simulations. Since the primary pocket of CRY1 is
targeted, coordinates of the side chain terminal carbon atom
of Arg358 (CZ in pdb file) can be used as grid center. Then, fine
tune the center by using the “Grid Options” panel. To visualize
the target pocket, the grid box panel should be used as follows:
Grid > Grid Box > number of points in x-dimension: 18;
y-dimension: 20; z-dimension: 20; x center: 11.7; y center:
35.7; z center: 16.4 (Fig. 3) (see Note 13).
3. Target pocket size and center information determined in step
2 will be provided in the configuration file that includes the
following keywords:
receptor, out, center_x, center_y, center_z, size_x, size_y,
size_z. Although these parameters are self-explanatory, options
that can be specified in AutodockVina are explained in notes
(see Note 14).
4. A sample configuration file named as “cry1_conf.txt” is
provided below:
24 Seref Gul and Ibrahim Halil Kavakli

Fig. 3 CRY1.pdbqt is visualized using AutodockTools. Grid box is colored in red-green-blue in x, y, and
z directions, respectively

receptor = CRY1.pdbqt
center_x = -11.7
center_y = 35.7
center_z = -16.4
size_x = 18.0
size_y = 20.0
size_z = 20.0

3.2.2 Preparation of Small molecule libraries from different companies are freely avail-
Small Molecules for able and can be downloaded from their websites. One of the
Docking Simulations commonly used libraries is ZINC library that has many tranches
classified according to properties of molecules such as molecular
weight and LogP. In addition to these physical properties, mole-
cules are under predefined categories, e.g., fragments, lead-like,
and drug-like.
1. Go to website: https://zinc.docking.org/tranches/home/
 Structure-Based Drug Discovery for Clock Proteins 25

Then click a tranch to download.

On the new page click # symbol on the top of the page and
select the format of molecules. We suggest downloading the sdf
format.
2. Rename the downloaded file to CRY1_sm.sdf. CRY1_sm.sdf
includes all molecules in one file. To extract molecules individ-
ually we are going to use the openbabel program. Type the
following to the terminal:

obabel CRY1_sm.sdf -O sm.sdf -m (see Note 15)

3. Molecules are in two-dimensional form. Openbabel (http://

openbabel.org/wiki/Main_Page) will generate three-
dimensional molecules with only polar hydrogen atoms in the
pdb format. For this type the following to terminal:

obabel sm1.sdf -O sm1.pdb --gen3D -d –AddPolarH

4. Next, to convert the pdb file to pdbqt format which includes

the charge information we are going to use the prepare_li-
gand4 module of AutoDockTools. Type the following to
terminal

~/MGLTools-1.5.6./MGLToolsPckgs/AutoDockTools/Utilities24/
prepare_ligand4.py -l sm1.pdb

This command is going to produce the pdbqt file with the

same name of the input pdb (see Note 16).

3.2.3 Running and Our receptor (CRY1.pdbqt) and ligands (sm1.pdbqt and others)
Analyzing AutoDock Vina are ready for docking simulations. We are going to use Autodock-
Simulations Vina software to run simulations. This program does not need to be
installed. Once the program is downloaded, giving a path to the
“vina” executable will be sufficient to run the program. “cry1_conf.
txt” was given in step 4 of Subheading 3.2.1. If all SMs and CRY1.
pdbqt is in the same folder,
1. Type following to the terminal:

~/autodock_vina_1_1_2_linux_x86/bin/vina --config cry1_conf.

txt --ligand sm1.pdbqt --out cry1_sm1_out.pdbqt --log
cry1_sm1_log.txt

2. Vina Binding Energy is printed on the screen. Also, output and

log file with the name given after --out and --log options,
respectively, are generated (see Note 17).
26 Seref Gul and Ibrahim Halil Kavakli

3. Docking simulations are completed now. Vina binding energies

can be sorted by using a python script available in the vina
manual page (http://vina.scripps.edu/manual.html) (vina_s-
creen_get_top.py).
On the terminal, script can be run as the following to get
the top ten molecules:

./vina_screen_get_top.py 10 (see Note 18)

4. We know our best molecules now. We are going to use Auto-

Dock Tools to analyze the results. Open AutodockTools
File > Read Molecule > CRY1.pdbqt > Open
Analyze > Docking > Open AutoDock vina result > (select
the best molecule) > Open
Select “Single molecule with multiple conformations” on
the “Load MODEL as” panel > OK.
To visualize the interacting residues:
Analyze > Macromolecules > Choose > CRY1 > Select
Macromolecule
Analyze > Docking > Show interactions (Fig. 4) (see Note 19)

Fig. 4 AutoDock Tools snapshot from analysis of docking results

 Structure-Based Drug Discovery for Clock Proteins 27

4 Notes

1. Providing “Project Title” will be beneficial for future analysis

since this query will be saved in the server with this entry. Some
searches for finding template(s) may take a couple of hours or
even days. When search is over, results will be notified through
“Email” provided.
2. Since novel molecules will be designed to PHR of CRY1,
crystal structures without ligand should be chosen for model-
ing. 4k0r, 5t5x, and 6kx4 PDB entries having the highest
coverage, 100% identity, and no ligand bound crystals can be
used to model full-length PHR. The 5t5x structure includes
residues between 1 and 496 found in the PHR of CRY1;
however, some residues in this domain are still missing.
SWISS-MODEL adds coordinates of missing atoms. The
C-tail of CRY1 is unstructured and is not resolved in 5t5x.
Thus, C-tail is also missing in our model. Overall, the model
includes only PHR of CRY1 that will be used for structure-
based small-molecule screening.
3. For each model SWISS-MODEL server provides quality scores
such as GMQE and QMEAN. GMQE is a score between 0 and
1 that is calculated based on alignment of target-template and
structure of the template. QMEAN is a Z-score that predicts
the nativeness of the structural features of the model. Basically,
it compares QMEAN scores of model and similar size crystal
structures. Scores below 4 is the indication of a poor model.
“Thumbs-up” or “Thumbs-down” symbol near to QMEAN
scores is the indication of “good” or “bad” model, respectively.
Some other quality controls and information of the model can
be found by clicking “\/” symbols on the right-hand side of the
panel.
4. Protonation states of ionizable amino acid residues such as
histidine, aspartic acid, and glutamic acid depend on the polar-
ity of the milieu and are quite substantial to design molecules to
the target pocket.
5. CHARMM forcefield is composed of topology and parameter
files. top_all36_prot.rtf file contains topology information for
proteins that is needed to generate protein structure file (PSF).
Topology file includes name, type, bond, and partial charges of
atoms in all amino acid residues. Parameter file includes force
constants for all bond-dependent terms and van der Waals and
nonbonded interaction terms.
6. PSF includes data for all molecules that enables the application
of a forcefield to the system. In this step a psf file for protein is
generated which is going to be used in the next steps. “psfgen.
in” has the following commands:
28 Seref Gul and Ibrahim Halil Kavakli

package require psfgen

topology top_all36_prot.rtf
segment U {pdb CRY1_p.pdb}
coordpdb CRY1_p.pdb U
guesscoord
writepdb CRY1_p_vmd.pdb
writepsf CRY1_p_vmd.psf

7. To run MD simulations, we need to solvate our protein. CRY1

should be placed in a box with 15 Å water layer in every
direction from the outermost atom in that direction. Then
add NaCl salt 150 mM as final concentration to mimic the
physiological environment. ionized.pdb and ionized.psf files
now include CRY1, water molecules and salt (NaCl) mole-
cules, ready for the MD simulation.
“solvation.in” has the following commands:
package require solvate

solvate CRY1_p_vmd.psf CRY1_p_vmd.pdb -t 15 -o CRY1_wb

package require autoionize

autoionize -psf CRY1_wb.psf -pdb CRY1_wb.pdb -sc 0.15

8. This command will measure the min and max coordinates in

x, y, and z directions. Our measurement gave the following
{52.63 10.58 79.04} {32.88 81.79 25.79}. These values
are going to be used to calculate the origin of the system and
vector length is needed in MD simulations.
9. restrain.in has the following commands:

set everyone [atomselect top all]

set fix [atomselect top “protein and name CA”]
$everyone set beta 0
$fix set beta 1
$everyone writepdb CA_rest.pdb

10. minimize.in can be found in supplementary materials. We

minimized the system by using conjugate gradient method
for 20,000 steps. Meaning of parameters used in MD scripts
can be found in this link: http://www.ks.uiuc.edu/Research/
namd/cvs/ug.pdf. Among many parameters, cellOrigin, cell-
BasisVector1, cellBasisVector2, and cellBasisVector3 are calcu-
lated from measurements given in Note 8. The arithmetic
average of x, y, and z coordinates is provided as the cellOrigin.
Extend of x, y, and z coordinates are calculated and provided as
(x 0 0), (0 y 0), (0 0 z) format for cellBasisVector1, 2, and
 Structure-Based Drug Discovery for Clock Proteins 29

3, respectively. The same cellBasisVector and cellOrigin coor-

dinates will be used in heating.in, equilibrium.in, and produc-
tion.in. After each MD step, log files should be checked for
error messages.
11. Configuration files heating.in, equilibrium.in, and production.
in can be found in the supplementary materials. In minimiza-
tion and heating steps canonical NVT (N, particles; V,
volume; T, temperature conserved) ensemble was used. In
equilibration and production simulations isothermal-isobaric
NPT (N, particles; P, pressure; T, temperature conserved)
ensemble was used.
12. By following the above steps non-polar hydrogens were
merged with atoms they bound and Gasteiger charges were
added to each atom.
13. Since the Vina program is using “Spacing (angstrom)” 1, set
this value 1.
x-center, y-center, and z-center values will be different for
each user because of the randomness in MD simulations.
We choose coordinates of Arg358 since mutation of
corresponding amino acid in CRY1 diminished interaction
with FBXL3 [36]. Pocket size is determined to cover the
whole primary pocket. PDBs are readable files, so coordinates
of the CZ atom in Arg358 can be read from a pdb file using a
text editor.
14. Following information is given in AutodockVina help menu:
Input:

--receptor arg rigid part of the receptor (PDBQT)

--flex arg flexible side chains, if any (PDBQT)
--ligand arg ligand (PDBQT)

Search space (required):

--center_x arg X coordinate of the center

--center_y arg Y coordinate of the center
--center_z arg Z coordinate of the center
--size_x arg size in the X dimension (Angstroms)
--size_y arg size in the Y dimension (Angstroms)
--size_z arg size in the Z dimension (Angstroms)

Output (optional):

--out arg output models (PDBQT), the default is chosen based

on the ligand file name
--log arg optionally, write log file
30 Seref Gul and Ibrahim Halil Kavakli

Misc (optional):

--cpu arg the number of CPUs to use (the default is to try to

detect the number of CPUs or, failing that, use 1)
--seed arg explicit random seed
--exhaustiveness arg (=8) exhaustiveness of the global search
(roughly proportional to time): 1+
--num_modes arg (=9) maximum number of binding modes to
generate
--energy_range arg (=3) maximum energy difference between the
best binding
mode and the worst one displayed (kcal/mol)

Configuration file (optional):

--config arg the above options can be put here

Information (optional):

--help display usage summary

--help_advanced display usage summary with advanced options
--version display program version

15. We assume that the openbabel program is downloaded and

installed properly. Using the command provided, each mole-
cule will be written to a single file, starting from the prefix
given after the -O option. File names will increase one by one,
e.g., sm1.sdf, sm2.sdf, sm3.sdf, etc. Openbabel program has
many options that can be seen by typing the following:

obabel -H

16. Full path to prepare_ligand4.py depends on where users

downloaded the MGLTools. “~” stands for the path of
home directory. After -l option, input is provided (in pdb
format) and pdbqt format is generated automatically with
the same name. All available options for this module can be
found by typing the following:

~/MGLTools-1.5.6./MGLToolsPckgs/AutoDockTools/Utilities24/
prepare_ligand4.py

To convert all pdb files to pdbqt for loop script can

be used.
17. To dock all molecules, a for loop script can be coded. Specify-
ing the number of CPUs will help to run docking simulations
 Structure-Based Drug Discovery for Clock Proteins 31

efficiently. For example, --cpu 8 option will allow programs to

use only 8 CPUs and the rest of the processors can be used for
other jobs. Unless --cpu is specified, Vina will try to use all
available processors. Another option that can be helpful to
mention is the exhaustiveness. While the default value of the
exhaustiveness is 8, increasing this value linearly increases the
computational time but exponentially decreases the probabil-
ity of not finding the minimum binding energy. Increasing the
exhaustiveness value, e.g., --exhaustiveness 12, will help to
improve your results especially if the target pocket is large.
However, computational cost should be taken into account
when screening a large library.
18. A few factors should be regarded to run this script. Script was
written for Python 2.7 or earlier versions; however, recent
Python versions (3 and newer versions) handle some variables
differently. Thus, to run this script without any editing default
python version should be considered. If multiple python ver-
sions were installed version can be specified to run script on
the terminal such as:

python2.7 vina_screen_get_top.py 10

Another point to mention is that the script is written such

that results were generated under a new folder. A quick
change in the script will allow us to analyze results generated
using the options given above. Change:

“file_names = glob.glob(’*/*.pdbqt’)” to file_names = glob.

glob(’*_out.pdbqt’)

19. Various options are available to visualize the molecule and

interacting residues. First one is “to update display for each
conformation.” Once it is clicked, which comes as the default,
as conformation of the molecule is changed by using arrow
keys, interacting residues will be updated automatically. Sec-
ond, to show molecules in surface representation that is the
default. Third, “close contact” atoms can be represented as
solid spheres or wireframes. Close contact residues can be
changed by playing with the “VDW Scaling Factor” option.
Another option is to show ribbon for “near residues” or
“for all residues.”
Labels on the contact amino acids can be shown or
hidden by clicking on “display labels on residues.” Pi-pi and
pi-cation interactions can be visualized, as well. Finally, the
generated favorite representation can be saved as a png file by
clicking on “Save Image.”
32 Seref Gul and Ibrahim Halil Kavakli
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Another random document with
no related content on Scribd:
stolen away under cover of the night and disappeared, rather than
face an investigation.
The daily papers had blazoned abroad the shooting of Randall
Batterly, and the subsequent trial of Wanza Lyttle, and my name had
appeared in the account, the writer who was my father’s lawyer
explained. A letter to the postmaster at Roselake had resulted in
further establishing my identity.
The writer had the honor to inform me that my father had left a snug
little fortune—the result of some recent fortunate mining ventures—
that would accrue to me, and he begged me to come back to my
southern home and take my rightful place among the people. I shook
my head at this. Who was there in the old home who would welcome
me? My mother was long since dead—my father gone. There was
no one belonging to me left in the old place. It would be more
strange and forlorn than an entirely new community. I should like to
visit it again. But that was all.
I dropped the letter to the floor, and sat thinking of Haidee. And as I
thought I smiled tenderly. After a time I decided that Haidee should
see these important letters—that I should go to her. And on a sudden
impulse I rose up.
As I opened the door the snow was falling, and there was a ring
around the moon. I left the door open and stepped back into the
house, going to the cedar room to get my sweater. When I returned,
a woman with snow-powdered hair was stepping hesitatingly across
the threshold. Haidee!
“It is you! Out so late—alone!” I began. “And in this storm.”
But the big eyes only smiled at me, and she stood there like a
beautiful wraith in her long gray cloak.
“Let me take your cloak,” I said.
I went to her, and she put both hands on my shoulders impulsively.
“I haven’t thought of the weather. Ever since I saw you last I’ve
thought of you,—and thought, and thought. It’s Christmas Eve, you
know. I have come to wish you a Merry Christmas, and I have
brought you a Christmas gift—one to keep till spring, at least.”
“Come to the fire,” I urged.
She sat down and I sat down opposite her. The firelight caressed
her, played in her eyes, ruddied her cheeks that were glowing from
her walk through the wintry air.
“In all the time I have known you this is the first time I have ever
shared your fire,” she whispered.
There was a silence. I could hear my heart-beats. How fine of her to
come to me in this womanly fashion! I sat and watched her. A lock of
hair had fallen over her ivory brow. She had dropped her head
forward on to her hand, and her dewy lips were parted. I stooped
closer, closer still. A tear slipped down on her smooth cheek and
glistened in the firelight as I gazed. She turned her face away.
“What gift have you brought me?” I whispered.
There was a movement in the shadows beyond the circle of light
cast by the green-shaded lamp—a rustle and a stir—then a swift
hurtling of a small lithe figure across the open space—a pause—a
swooping, frantic clutch of young strong arms about my neck, and
Joey, all wet and steaming in his snowy coat, had me fast, shouting
in my ear, over and over again:
“I’m your Christmas gift, Mr. David! I’m your Christmas gift.”
He was in my arms, and Haidee had drawn back and was smiling at
me, her eyes like great luminous pools of fire.
“What a wonderful, wonderful present,” I responded shakily. “Now,
who could have sent me this very best present in the world?”
“Bell Brandon,” shrieked my little lad. “She did not send me—she
brought me.”
“Then—she must have another gift for me,” I said boldly, and held
out my hand to Haidee.
She shook her head, her eyes grave, but her lips still smiling.
“I have brought Joey to you—but—I cannot stay. I am going away.
Will you keep my boy until I return?”
“You are going away?”
She bent her head.
“I am going to take Wanza back East. I want to go away for a time—
it is best for me to go. But—you must not be separated from Joey all
this long winter, David Dale. My boy shall stay with you—and in the
spring I shall come for him—or come back to stay at Hidden Lake.”
“You are going away—soon—after Christmas?”
“To-morrow. We are going to-morrow—Wanza and I—we decided it
only to-day. I have some matters to attend to in New York. I must go
at once.”
“Christmas Day?”
“Yes.”
“Wait—do not go—stay with me as my wife, my wife! I have sold my
book—I am free too, of an old, old shadow. Oh, I have much to tell
you—much to talk over with you. Wait—let me read to you some
letters.”
My voice was rough with emotion. She held up her hand.
“When I come back, David Dale, my friend—not now. We need to
gain perspective—you and I. I have been through an ordeal—I am
shaken—I am not myself. I don’t see clearly. And as for you—David
Dale, there is much for you to learn.”
“What do you mean?” I cried brusquely.
She smiled at me sweetly and a little sadly.
“Oh, you are a stupid blundering David.” She shook her head. “But—
wait till spring.”
“There is so much I want to say—explain,” I stammered.
“Wait till spring.”
“But I cannot keep Joey. I cannot let you go without your boy.”
“He will be better off with you.”
“I cannot accept such a sacrifice.”
On this point I remained firm. We argued. Haidee entreated, and
Joey begged to be allowed to stay. I would not listen to either voice. I
arose at last.
“Joey,” I said, speaking slowly, in order to steady my voice, “I have
one more bolt to put in the sled I am making for you. Will you come
to the workshop with me?”
And in the shop away from every eye, I said good-bye to my lad. And
as I kissed him the old doubt stirred. Was I so sure he was Haidee’s
child?
Old Lundquist came for Haidee; and we said a conventional good-
bye beneath his prying eyes.
Until twelve I waited and watched for Wanza, expecting every instant
to hear Captain Grif’s voice at the door, and to see Wanza step over
the threshold. Surely she would not go without some last word to me.
But she came not.
 CHAPTER XXIV
“THE FLOWER WILL BLOOM ANOTHER YEAR”

I SAT by my fire throughout the long night. When dawn came I rose,
went to the door and threw it wide and stepped outside into the
unstained air of the morning. There was a carpet of snow on the
ground, the bushes were like gleaming teepes, and the limbs of the
pine trees were weighted with icicles. I repeated to myself Thoreau’s
words: “God exhibits himself in a frosted bush to-day, as much as he
did in a burning one to Moses.”
The light was purple and cold and solemn, the moon still hung in the
gray of the western sky, but in the East there was a glorious band of
crimson and the mountain tops looked as if aflame with little bonfires.
As I stood there a ruby-crowned kinglet fluttered from twig to twig of
the elderberry bush hard by, emitting its bright “zei, zei,” and a
chickadee answered with a merry “chickadee-a-dee, dee, dee,” from
the yew grove. I waited. I was praying the kinglet would sing. And
presently the tiny thing began. It poured forth its strong sweet notes
in a succession of trills.
“Bird,” I said, “you are a wonder. I know that the muscles in your
throat are almost microscopic. I have always told Joey—” But here I
ceased to admonish the bird, I went back up the porch steps.
As I was closing the door I heard the rattle of the stage as it passed
along the river road on its way to the village. The driver shouted a
merry Christmas to some one on the road. I threw a fresh log on the
fire and sat down heavily in my chair. It was Christmas morning—
and they had gone!
I drowsed after a time, lying back in my great chair with the collie
asleep at my feet. When I awakened the sun was high, and the world
outside my window was so sparkling and bright that it dazzled my
sight. I went to the kitchen, kindled a fire, and opened the kitchen
door to let the collie out. I was washing my hands at the wash-bench
in the corner, when I heard the latch of the door click. Footsteps
crossed the floor, some one was coming up behind me saying:
“I have brought a chicken pie for your dinner, Mr. Dale—Dad’ll be
along soon—and I wish you a Merry Christmas.”
It was Wanza.
She stood there as she had so often stood before, a white-covered
basket on one arm, the other filled with bundles. But her face was
pale to-day, and her glorious hair was swept straight back from her
brow and tucked away beneath a net, and her apparel was sober
gray. I stared at her and stared and stared, until the pink ran up in
her cheek and she dropped the bundles and set down the basket,
that she might put her hands over her abashed face. I stood there
and felt shaken and dumbfounded, not attempting to speak, afraid
indeed of the sound of my own voice.
The fire crackled. Cheerily through the door Wanza had left open
behind her, came the chickadee’s note. The sunlight was dazzling as
it struck into my eyes from the white oilcloth on the kitchen table. The
room seemed suddenly illumined, the air electric and revitalized. At
length I stammered out:
“Thank you, thank you!”
“It’s only chicken pie,” she whispered.
“Thank you for not going.”
At that she threw up her head, her hands dropped. She said proudly:
“Did you think I’d go on Christmas Day? Did you think I’d have the
heart to go, Mr. Dale?”
“Yes,” I said wearily, “I thought you had gone, Wanza. Why not?”
“And I’ll tell you why not! It’s because you decided Joey was to go
that I could not go. I could not go and leave you when I found Joey
was to go—oh, no!”
“But you must go some day, Wanza,” I said, scarce knowing what I
said.
“And why must I go some day? Why must I? I tell you what I’m going
to do, Mr. David Dale, I’m going to stay on here in Roselake, and I
am going to live up to the very best there is in me. I am going to
improve and grow big and fine and womanly. I’m going to do it right
here. And then maybe some day,” she sighed, “when Dad does not
need me any more, and you do not need me any more, I will have
enough money saved up, and I will go away and get educated.”
In her excitement she had pressed closer to me and laid one hand
against my chest. I placed my own hand over it as I said very gently:
“Let me teach you, Wanza—be my pupil. I will become your tutor in
earnest, if you will have me. Yes! I will go to your father’s house
every day to instruct you,—and it will give me great happiness. Ah,
Wanza, now that Joey has gone I feel so futile—so useless! Let me
undertake your education, child.”
The burning eyes came up to mine, and questioned them. The pale
face flushed. There was a pathetic tremulousness about the lips.
“Say yes,” I urged.
Her head drooped, lowered itself humbly until her hair brushed my
arm, and suddenly she kissed my hand, passionately, gratefully. “Oh,
Mr. David Dale,” she breathed, “you’re grand! That’s what you are.
Yes and yes, and yes!”
And so I ate my dinner with Wanza and Captain Grif sitting opposite
me at the table, and Wanza flouted me when I would have served
her too liberally with the most succulent bits of the pie, and Captain
Grif rallied me when I confessed that I had small appetite, and
produced a bottle of root beer and a bag of cheese cakes from the
basket.
Night came down at last to my weary soul and soon after it grew
dark Wanza and her father departed. I locked the door behind them
and I threw myself, dressed as I was, on my bunk and buried my
head in the pillows. The evening wore on. The fire sputtered and
burned low, the wind came up and hissed around the cabin. A coyote
howled from some distant hill. The room grew dark. A pall was on my
heart.
As the winter wore on I became vastly interested in Wanza’s
education. I gave two hours each day to her lessons. And not many
evenings passed without lessons in the snug little room beneath the
eaves of the cottage she called home. There with our books open
before us, beneath the light from the swinging lamp, we pored over
tedious pages shoulder to shoulder, smiled on by old Grif and
encouraged by Father O’Shan, who ofttimes shared our evenings.
It was wonderful the improvement I marked in Wanza as the weeks
slipped past. Her English improved markedly. She was painstaking
and indefatigable. She applied herself so assiduously that I began to
fear lest she should overwork, as the warm spring days came on.
“Don’t study too hard,” I cautioned her one day.
“I can’t study too hard,” she flashed back at me. And then she
smiled. But I knew she was terribly in earnest.
It was that same day that Father O’Shan quoted to me, as we were
walking along the river road together:
“Shed no tear—Oh, shed no tear!
The flower will bloom another year.
Weep no more—Oh, weep no more!
Young buds sleep in the root’s white core.”
“Do you mean that for me, Father?” I asked.
“For you—yes. And many like you.”
My heart swelled. I looked about me. Buttercups were gilding the sod
—the pussy willows were in bloom along the river. It was the spring.
I went home and raked the dead leaves and pine needles away from
under the trees in the Dingle. A few yellow violets were springing up.
From beyond the syringa thicket a faint “witchery, witchery, witchery,”
greeted my ears.
I went forward cautiously. Peering through the interlaced branches I
saw the songster. He was swinging on a thorn bush, a wonderfully
brilliant little chorister in his black cap and yellow stole. I whistled. He
cocked his head on one side, fixed me with his bright eye, then flew
to a willow tree and favored me with another burst of song. This time
he seemed to oft repeat, “Which way, oh?” He sang it so persistently
that presently I replied, “Straight on, sir.”
I went to the cabin and consulted the calendar. It was the last day of
March.
My spirit, that had seemed earthward crushed for months, grew
lighter in the sweet spring days that followed. I took the return of
April as a long-fore-gone right. I ploughed and planted, I made bird
houses and arranged bird-baths in the groves hard by the cabin. I
paddled in my canoe on the river, and fished in the adjacent creeks.
And I went with Wanza through the woods on many a trillium hunt.
Sometimes almost to breathlessness I felt Wanza’s charm, the
galvanism she could always transmit to those with her intensified by
some new strange quality I could not name. It was like a fillip given
my dispassion. When she laughed and chirped to the squirrels, when
she carried a wounded bird in her breast, when she stood on tip-toe,
her face like a taper-flame, to greet the whole outdoors with wide-
flung arms, I caught my lip between my teeth and watched her with
observant eyes. Her beauty grew. Even Father O’Shan remarked it.
The gowns of pink she wore once served to deepen the rose tint in
her fair cheeks; but her cheeks needed no such service now; they
were like a red-rose heart. She had taken to smoothing and banding
her hair and twisting it back behind her small ears with big shell pins.
Her head seen thus was as lovely a shape as any Greuze ever
painted. She frequently wore thin blouses of white, and I seldom saw
her feet in sandals—she had a sleeveless black gown that she wore
to a country dance one evening when I was her escort. Looking at
her that night I could scarcely believe it was Wanza, my old friend
and playmate whom I was in attendance upon, and I paid her some
rather silly compliments and was promptly rebuked for my gallantry.
It was a tidy enough fortune my dear old father had left me. I had
been able to do many things to make Wanza and Captain Grif
comfortable and happy during the long winter. Among other things I
had purchased a piano for Wanza to replace the old melodeon, and
delighted Captain Grif with the gift of a phonograph. And last, but not
least, I had made the last payment on the little cottage in which they
lived and presented the deed to Captain Grif on his sixty-fifth
birthday.
Dear Captain Grif! His manner of accepting this last gift was
characteristic.
“Tain’t for myself I’d take it. I’d just about as lief worry along and save
and scrimp toward makin’ the final payment— I ’low I’d sooner; I like
the glory, and when you have a soft thing handed to you there ben’t
nothin’ achieved. I’m meanin’ it, s-ship-mate. Things we earn is the
things we ’preciate. But I take it kindly of you. And for Wanza’s sake I
thank you and accept. ’Tis hard on the gal—pinchin’ and scrimpin’—
and peddlin’ in winter is about played out—the roads is in bad shape
for gettin’ about, you’ll ’low. Now with the house paid for, the gal’ll
have what she earns for ribbons and furbelows and trinkets. And
ownin’ sech a face as hern, Mr. Dale—though it don’t need no
adornin’—sure makes a gal long for fixin’s. I’m grateful and pleased
for her sake—I sure be.” Tears dimmed his kind old eyes. His hand
came out to me. “Shake hands, David Dale, man; you’re a friend—a
friend. We need friends—the gal and I—seems like we need ’em
more’n we used since all we been through,—and I want to say right
here that Wanza never would’a perked up if it hadn’t a been for your
helpin’ her this winter. She was pretty well down, Wanza was. Well,
in my youth, young folks was different. I used to think—I used to
think one time—well, there, by golly, s-ship-mate, it makes no
difference what I used to think! I was mistook, I ’low. It sure is great
for a man and gal to be such friends as you and Wanza—no
foolishness—no tomfoolery!—it’s unusual—I ain’t sayin’ that it tain’t
—but it’s fine, s-ship-mate, it’s fine.”
 “I’M GRATEFUL AND PLEASED”
Through the winter I had had frequent letters from Haidee—frank,
friendly letters, filled with stories of Joey—and a few printed epistles
from the lad; one in particular that impressed me; “Joey is all rite,” it
said.
I discussed this with Wanza, who said tearfully:
“His saying that makes me think he isn’t. He is such a plucky little
chap. He would not have you worrying. Not that I think he’s sick—
sure enough sick, you know; but I just feel sure he’s pining.”
“Please—please, Wanza, don’t put that thought into my mind,” I said
hastily. “If I thought Joey were happy I could more easily bear his
absence.”
She looked at me and shook her head. Then she smiled.
“He’ll do well enough till spring. But he will be counting the days, all
right.”
 CHAPTER XXV
MY SURPRISE

WHEN May came I began to look forward in earnest to the return of

Haidee and Joey. Every day since the beginning of spring I had gone
to Hidden Lake to tend the vines and shrubs that I had set out with
so much care the previous fall. I had also made a flower bed and
planted the seeds of many old-fashioned flowers—larkspur, Sweet
William, marigolds, phlox, lobelia, clove pinks and mignonette, sweet
peas and rosemary. In another few weeks the little cabin would be
surrounded by bloom.
A Vigor’s wren was building a nest in the pergola, and a calliope
humming-bird’s nest hung on a pine limb near the kitchen door, not
more than eight feet above the ground. I could scarcely wait for Joey
to see the latter. The hours I spent at Hidden Lake were filled with
strange anticipations, and unanswered questions and grim
wonderment.
But Fate had a surprise in store for me.
One day as I stood looking at the humming-bird’s nest a man
approached the cabin from the wood path beyond the garden. He
was a hard-faced man, a grizzled, uncouth figure of a man. I took an
instant dislike to him without even waiting to see his features. When
he saw me he halted irresolutely. I nodded to him carelessly, and
stooped to pull a stray weed from the bed of thyme beside the
kitchen door. When I looked up he stood beside me.
“Good day, sir,” he said.
“Good day,” I returned.
“Is Mrs. Batterly to home?”
“No,” I replied, “Mrs. Batterly is in the East.”
“Is her cabin shut up?”
“It is,” I said curtly.
“Well, I swan! Say, did she take the kid with her?”
“She took the little boy with her, certainly.”
He grinned, showing blackened teeth and unsightly gums. “Um,” he
said, half shutting his red-lidded eyes, “um, um—you’re Mr. Dale, I
take it; I have seen you in the village.”
“Yes, I am David Dale,” I answered straightening up. “Is there
anything I can do for you?”
He guffawed. “No,” he chuckled, “you can’t do a darn thing for me,
but you bet your gosh darned boots I can do something for you.”
I turned away in disgust.
“Say, partner,” he pulled me round to him by the sleeve, “I reckon
that Mrs. Batterly took the kid with her thinking the kid was hern.
Well, he ain’t!”
I gaped at him. He grinned at me in a would-be friendly manner.
“My name’s Bill Jobson. I’m a miner,” he volunteered.
“That means nothing to me,” I told him sharply.
“Well, now, I don’t suppose it does! See here! I’m the man as helped
Randall Batterly kidnap your boy, Joey— Wait a minute, wait a
minute! Don’t get excited. It was a frame up—the whole darn thing!
Batterly never had no idea the kid was his. He framed the whole
thing up to get a rise out of his wife. He was set on getting her back,
and he took that way of doing it. He knew mighty well the kid warn’t
his. His own boy died from an over-dose of medicine Batterly gave it
one night when he was drunk, on board the ship him and me was on
going from Alaska to Seattle. The boy died in my arms, and was
buried at sea. Batterly wouldn’t go back to Alaska and face his wife
and tell her the truth about the child. He made me swear not to
squeak. And he went back, and he let on to his wife that the child
was never seen after the collision between our ship and another, in
the fog, off Cape Flattery. He told his wife as how a nurse on board
ship had the babe in her stateroom, caring for it, the night of the
wreck. There was a nurse on board who was drowned that night, so
the story passed muster.”
I watched the man with fascinated eyes as he sat down on the
doorstep, filled his pipe leisurely, and struck a match on his boot
heel. The full import of his statement did not sink into my brain at
once. When it did I said, speaking with dry lips:
“But what about the mark on the lad’s chest?”
“That’s what you call a coincidence, partner—that and their age
seeming to be the same. When Batterly saw the mark on the kid’s
chest the whole blame plan came to him quick as lightning, he said.
And when the girl, Wanza Lyttle, told him as how he was picked up
by a fisherman over on the Sound, that settled it. He took a chance
on his wife’s not remembering the mark on her kid’s chest was just
over his heart. This kid’s is higher up.”
Completely unmanned, I sat down on the step beside my visitor, and
rested my head in my hands. “It does not seem possible your story is
true,” I groaned.
Bill Jobson brought his hand down hard on my knee. “Look ahere,
Mr. Dale, do you think I tramped way over here from Roselake to see
Mrs. Batterly just because I wanted a country stroll? Well, I didn’t!
Get that through your head—quick! I’m a busy man— I oughtn’t to
have took the time to come and say my say as I have—”
“Will you write a statement and have it witnessed, and send it to Mrs.
Batterly?” I interrupted.
“I will that. And I’ll tell you why I’m doing it. I’m doing it because I
used to see the little chap with you in the village last summer and I
saw him after that in the fall with Mrs. Batterly, and he never run and
skipped as he did with you. It just got me for fair—it did! I’ve been
intending all this winter to see Batterly’s widder and tell her the gosh
darned truth, but I been working in the Alice mine, a good fifty mile
from Roselake, and I ain’t been down but once before since fall, and
that time I—well, I got pickled, partner, I sure did! I wa’n’t exactly up
to holding lucid conversation with folks, you might say.”
I was silenced.
That night the statement was written in the presence of Captain Grif,
Wanza, and Father O’Shan, and it went forward with a letter from me
to Haidee.
Wanza and I waited impatiently for a return letter from Haidee. But
the days went past like shadows, and no letter came. I had been
climbing upward toward the summit of comparative peace, I had
almost reached it when Bill Jobson came with his disclosure. But
now, hearing nothing from my wonder woman, the valley closed
around me. I walked in a stagnant marsh, the atmosphere was that
of the lowland.
One night some three weeks after the letter from Haidee should
have reached me, I found myself unable to sleep. I arose and
dressed, and went outside and walked along the river road toward
the village. After going some distance I lay down beneath a tree in a
pine grove. It was about two o’clock. A purple darkness lay all
around me. The stars were like pale gems, clear and cool and
polished. The Milky Way was like a fold of silver gauze. The pines
stood up very black and silent in my grove. I began to wonder why I
ever slept indoors, when out in the woods I felt as though I were in
God’s house, a partaker of his hospitality.
I relished my bed of pine needles extremely. I began to ponder many
things, the silence and the stars served to give my thoughts a
strange turn, and I recalled what a well-loved writer has said: “To live
out of doors with the woman a man loves is of all lives the most
complete and free.”
Yes, I said to myself:
“Wandering with the wandering wind,
Vagabond and unconfined.”
Slowly I said over to myself the last verse of the song—the verse I
had not given to Wanza:
“Marna of the far quest
After the divine!
Striving ever for some goal
 Past the blunder-god’s control!
Dreaming of potential years
When no day shall dawn in fears!
That’s the Marna of my soul,
Wander-bride of mine!”
Wander-bride of mine! Was it a woman like Haidee who had
suggested those lines to the poet?— Haidee with her narrow, oval
face, and brow of ivory, and slow, bell-like voice. Or had it been
some elf-girl, some girl of flame with a temperament wilder than most
—a gipsy thing of changing moods, and passionate phases of self-
will, alternating with abnegation and tenderness,—with a face like a
wind-blown flower, and a nature very human, very lovable and rare!
—a girl like Wanza—say?
After a time I slept. When I awakened the horizon showed a silvery
light. The purple darkness still mantled the woods and the stars still
shone, but day was coming on apace. As I lay there, half dozing, and
gradually becoming tranquil and restored, I heard faint footfalls and a
modulated whistling on the road beyond. There was a mellowness
about the whistle that was infinitely piquant, some quality that stirred
me as a bird’s song stirs. Doubtless some ranch hand thus early
astir, I said to myself.
I had not long to speculate, for the whistler approached, left the road,
and entered the grove wherein I lay. I could hear a light crackling as
the invader of my solitude brushed through the growth of young
scrub pines. The whistle changed to a low song, and the song was
sung in a woman’s voice.
It was Wanza who was coming through the pines toward me!
When she was comparatively near I spoke from my couch beneath
the tree.
“Hist! Hist! Wanza!”
The song ceased. I knew she was standing stock still.
“Who—who—where are you?” her voice sounded frightened.
“I’m David Dale. And I’m not ten feet from you—follow my voice.
Don’t trip on the tree roots.”
She came towards me slowly. I stood up and went to meet her. As I
advanced a strange glee took possession of me. I was elated at this
unexpected encounter, this beautiful rendezvous between darkness
and dawn in the pine forest. And at the thought of a companion to
watch with me the coming in of day.
I took her hand silently. We went forward to the pine tree and sat
down together beneath it. Wanza did not speak. I was enchanted
because she did not. I could just dimly see her face. Her head was
thrown back, and I knew her eyes were lifted.
The light began to spread over the east. Soon the mountain tops
were touched with orange fire. A cool breeze sprang up, and the
young hemlocks on the hillsides swayed and tossed their fringes. But
the pines in our grove stood immovable and black, and the wood
vistas were unlit. I heard the river, and the babble of a rillet in a draw
hard by. The dulcet sounds were the only sounds we heard. The
whole world seemed waiting. We sat thus for perhaps ten minutes,
while the light spread over the east and the purple darkness of our
grove gradually gave way to a cool gray aspect. And then the sun
came up, a spurt of liquid amber in the urn of the sky, and its light
trickled far out over the hills, and the stars grew pale and
disappeared. The day had come.
I was exhilarated. I was filled with full measure of good will and
gratification. And I glanced at my companion, to read in her face her
appreciation of the miracle. She was smiling ineffably, and as I
turned fully towards her, she closed her eyes. I became conscious
then that I was holding out my hand to her. I looked down at it
curiously, and I looked at her face, bent forward and peered at it
again. Who was this companion who had shared my solitude, and by
her understanding made it perfect?—who had given me quiet
fellowship, sat near me in the starlight, watched the day come in with
me, and now rested within reach of my hand? Who should it be, I
answered myself wonderingly, but my old friend and companion,
Wanza?
She opened her lids and I saw the wonder of the sunrise in her eyes,
and something mysterious and deep blended with the languor of
sleep. And when she smiled at me and whispered my name, I
quivered suddenly and the blood surged unbidden into my face.
“Wanza,” I said, “Wanza!”
“Yes?” she breathed.
“Hasn’t it been wonderful, Wanza? Hasn’t it been miraculous? ‘Every
hour of the light and dark’ is a miracle, but the sunrise is the greatest
one of all. It is arresting. I can never drop off to sleep again if I waken
and see the sky rosy.” I spoke with a fluttered haste, my words
tumbling over each other in a way not at all characteristic, and when
Wanza whispered: “Why, neither can I,” I laughed outright joyously.
“I found a wonderful wake-robin in the woods yesterday,” I began
after a pause; “the petals were pink and strongly veined, and it was
monstrous—monstrous! petals two inches—well, almost two inches.
It must be a large-flowered wake-robin. The trilliums have been
profuse this spring. This fellow was belated—its companions are all
gone.”
“The robins woke up two months ago,” Wanza said, shyly eager.
“And they have finished their courting.”
“Yes, they are very wide-awake, and business-like. But they have not
finished their courting,—I am sure I witnessed a love scene
yesterday.”
“Not really, Mr. Dale?”
“It looked uncommonly like one.”
In the growing light I saw that her face had kindled. It was lifted to
mine, and she was drinking in every word. The emotion the sight of
that kindled face aroused in me started a train of thought, and
checked the words on my lips. Oh, in very truth there was something
puzzlingly complex about my feeling for Wanza! I recoiled as from
some revelation that I did not care to face as she continued to smile
at me. But her eyes drew me, and I leaned forward and peered into
them; and as once before I read their message, but I continued to
gaze this time until the lashes swept down and the light was hid.
I walked back to the village with Wanza, and there was the tinkle of
bells on cattle awake in the meadows, and the stir of sheep milling
on rocky hillsides, and the crowing of cocks and the chirp of birds to
proclaim that morning had come. We were almost at the village
when she put a question to me.
“Mr. Dale, do you know what day to-morrow is?”
I had been expecting the question and dreading it.
“Yes,” I answered, “I know well that it is the day we have been
accustomed to celebrate as Joey’s birthday.”
I spoke impatiently. But when I saw the tears in her eyes, I stopped
there in the road and took her by the shoulders and turned her
around to me ruthlessly, crying:
“Listen to me! You must be hurt, if you will, at my surliness, Wanza
Lyttle! I cannot keep my tongue smooth when my nerves are ragged.
We go on and on, and bear much—stoically—for weeks, months,
years, indeed, and then—suddenly, we can bear no more! We reach
the pinnacle of pain. We cry out—with the poignancy of it. But after
that, I have a fancy, we can never suffer so much again. I am at the
pinnacle. There is no last straw for me. It has been placed. After to-
morrow the worst will be over. God! let me get through the day and
play the man.”
She said not a word. We parted silently. But after I had gone a little
way she came running after me.
“I only wanted to say, David Dale,” she breathed, “I only wanted to
say—”
“Yes, Wanza?”
“I only wanted to say, ‘God bless you.’”
 CHAPTER XXVI
THE OLD SWIMMING HOLE

AND so I came to the day that was sacred to Joey!

I began it by ploughing in the field back of the cabin. I went not near
the shop. I did not venture into the cabin for lunch at noon. I had
made up my mind to work doggedly till sundown and then go to the
village inn for supper, and later join Father O’Shan at Captain Grif’s.
Someway it comforted me to think of the evening; of the snug little
nook beneath the eaves; and of the welcome that awaited me there.
I saw Wanza’s face, in fancy—solicitous, pleased; I saw her figure
there in the centre of the room, clasped by the yellow light of the
swinging lamp, her hair gilded by its rays, on her cheek an eager
flush. Kind heart! Dear, helpful girl! Cheerful, buoyant, valiant little
wander-friend!
The sun for a June sun was unduly fervid, so that by four o’clock I
was weary and dripping with perspiration, and longing for a dip in the
river. I rested, and leaning on my plough, looked away through the
cedars and cottonwoods to the green of the river flashing in the
sunlight. I heard the rattle of the stage on the road, and when I was
certain it had passed I went to the cabin and put on my bathing suit. I
went in at the back door of the cabin, and out at the front, passed
through the yew grove, crossed the bridge to the shop, and so
gained the river bank and my favorite swimming hole beneath the
cedar trees.
The spreading trees threw a deep shade over the pool. It was almost
twilight beneath their network of branches. And I was on the bank
prepared for a dive before I saw a small figure below me seated on a
boulder at the edge of the water, half hidden from view by the steep
slope of the bank. I saw the flash of bare feet in the water. Poised